The present invention relates to substantially pure antiviral biflavanoids, e.g., robustaflavone, biflavanoid derivatives and salts thereof such as esters, ethers, amines, sulfates, ethylene oxide adducts, and acid salts, and pharmaceutical compositions containing the same. Representative examples include hexa-O-acetate and hexa-O-methyl ether derivatives of robustaflavone and robustaflavone tetrasodium salt. The present invention also relates to methods for extracting substantially pure robustaflavone from plant material. The present invention also relates to a method for preventing and/or treating viral infections such as hepatitis B, influenza A and B, and HIV which employ robustaflavone or derivatives thereof alone or in combination with at least one antiviral agent such as 3TC.
Viruses, an important etiologic agent in infectious disease in humans and other mammals, are a diverse group of infectious agents that differ greatly in size, shape, chemical composition, host range, and effects on hosts. After several decades of study, only a limited number of antiviral agents are available for the treatment and/or prevention of diseases caused by viruses such as hepatitis B, influenza A and B and HIV. Because of their toxic effects on a host, many antiviral agents are limited to topical applications. Accordingly, there is a need for safe and effective antiviral agents with a wide-spectrum of anti-viral activity with reduced toxicity to the host.
Since the identification of the human immunodeficiency virus (HIV) as the causative agent of AIDS,36,46 the search for safe and effective treatments for HIV infection has become a major focus for drug discovery groups around the world. Investigations into the molecular processes of HIV have identified a number of macromolecular targets for drug design, such as HIV-1 reverse transcriptase (HIV-RT), protease and integrase enzymes, and regulatory proteins (e.g., TAT and REV). Other targets are enzymes which aid in virus attachment and fusion. HIV-RT is an essential enzyme in the life cycle of HIV, which catalyzes the transcription of HIV-encoded single-stranded RNA into double-stranded DNA. Furthermore, the RNA-dependent DNA polymerase function of HIV-RT does not have an analogous process in mammalian metabolism, and thus is a suitable target for a chemotherapeutic agent.
The hepatitis B virus (HBV) infects people of all ages. It is one of the fastest-spreading sexually transmitted diseases, and also can be transmitted by sharing needles or by behavior in which a person""s mucus membranes are exposed to an infected person""s blood, semen, vaginal secretions, or saliva. While the initial sickness is rarely fatal, ten percent of the people who contract hepatitis are infected for life and run a high risk of developing serious, long-term liver diseases, such as cirrhosis of the liver and liver cancer, which can cause serious complications or death.1 The World Health Organization lists HBV as the ninth leading cause of death. It is estimated that about 300 million persons are chronically infected with HBV worldwide, with over 1 million of those in the United States. The Center for Disease Control estimates that over 300,000 new cases of acute HBV infection occurs in the United States each year, resulting in 4,000 deaths due to cirrhosis and 1,000 due to hepatocellular carcinoma.2 The highest rates of HBV infections occur in Southeast Asia, South Pacific Islands, Sub-Saharan Africa, Alaska, Amazon, Bahai, Haiti, and the Dominican Republic, where approximately 20% of the population is chronically infected.3 
Hepatitis B virus (HBV) infection is currently the most important chronic virus infection, but no safe and effective therapy is available at present. The major therapeutic option for carriers of HBV is alpha interferon, which can control active virus replication. However, even in the most successful studies, the response rate in carefully selected patient groups has rarely exceeded 40%.5,6 One of the reasons cited for interferon failure is the persistence of viral supercoiled DNA in the liver.7 
Recently, lamivudine (3TC) has provided encouraging results against both HBV and HIV in human clinical trials. Lamivudine is approved for treatment of HIV infection, and is currently being evaluated as a treatment for HBV. A recent study of 40 HIV-HBV-coinfected patients showed dramatic decreases in the level of HBV replication following treatment with 3TC over a 12 month period, with no observable adverse effects (84). Another agent, famciclovir, an orally active derivative of the acyclic guanine derivative penciclovir (85), is approved for treatment of herpes zoster and acute recurrent genital herpes, and has also shown promising results against HBV infection in clinical trials, again with little observable toxicity (86). It is hoped that these agents will eventually provide promising treatment options for HBV infection.
Unfortunately, monotherapy of viral infections often results in selection for mutant viral strains having resistance to the antiviral drug being used. Indeed, clinical isolates of mutant HBV strains have been identified having resistance to 3TC following treatment with that agent (87,88). It is plausible that combination therapy of HBV would provide an enhanced antiviral response, while reducing the danger of resistance selection. Combination of agents has been shown to be superior in treatment of HIV relative to monotherapy (89). Thus, the identification of compounds which inhibit HBV, particularly compounds having structures other than that of the nucleoside analogues, is of critical importance in the search for effective anti-HBV regimens.
Influenza is a viral infection marked by fever, chills, and a generalized feeling of weakness and pain in the muscle, together with varying signs of soreness in the respiratory tract, head, and abdomen. Influenza is caused by several types of myxoviruses, categorized as groups A, B, and C4. These influenza viruses generally lead to similar symptoms but are completely unrelated antigenically, so that infection with one type confers no immunity against the other. Influenza tends to occur in wavelike epidemics throughout the world; influenza A tends to appear in cycles of two to three years and influenza B in cycles of four to five years. Influenza is one of the few common infectious diseases that are poorly controlled by modem medicine. Its annual epidemics are occasionally punctuated by devastating pandemics. For example, the influenza pandemic of 1918, which killed over 20 million people and affected perhaps 100 times that number, was the most lethal plague ever recorded. Since that time, there have been two other pandemics of lesser severity, the so-called Asian flu of 1957 and the Hong Kong flu of 1968. All of these pandemics were characterized by the appearance of a new strain of influenza virus to which the human population had little resistance and against which previously existing influenza virus vaccines were ineffective. Moreover, between pandemics, influenza virus undergoes a gradual antigenic variation that degrades the level of immunological resistance against renewed infection.4 
Anti-influenza vaccines, containing killed strains of types A and B virus currently in circulation, are available, but have only a 60 to 70% success rate in preventing infection. The standard influenza vaccine has to be redesigned each year to counter new variants of the virus. In addition, any immunity provided is short-lived. The only drugs currently effective in the prevention and treatment of influenza are amantadine hydrochloride and rimantadine hydrochloride.11-13 While the clinical use of amantadine has been limited by the excess rate of CNS side effects, rimantadine is more active against influenza A both in animals and human beings, with fewer side effects.14,15 It is the drug of choice for the chemoprophylaxis of influenza A.13,16,17 However, the clinical usefulness of both drugs is limited by their effectiveness against only influenza A viruses, by the uncertain therapeutic efficacy in severe influenza, and by the recent findings of recovery of drug-resistant strains in some treated patients.18-22 Ribavirin has been reported to be therapeutically active, but it remains in the investigational stage of development.23,24 
In influenza, amantadine and rimantadine have been shown to be moderately effective against only influenza A viruses; with amantadine having excessive side effects. Recently, strains of influenza A resistant to amantadine and rimantadine have been isolated. Accordingly, there is a need for new types of therapeutic antiviral agents against both influenza A and influenza B, as well as against HBV and HIV.
The present invention relates to substantially purified antiviral biflavanoids, derivatives and salts thereof and pharmaceutical compositions containing the same; improved methods for extracting substantially pure robustaflavone from plant material; methods for preparing derivatives and salts from antiviral biflavanoids; and methods for treating and/or preventing viral infections using the antiviral biflavanoids, derivatives and salts thereof.
The present invention provides substantially purified biflavanoids comprising robustaflavone, hinokiflavone, amentoflavone, agathisflavone, morelloflavone, volkensiflavone, rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a and pharmaceutical compositions containing the same. Scheme I illustrates the chemical structures of these biflavanoids. The biflavanoids of the invention, extractable from plant materials derived from a variety of natural sources such as Rhus succedanea and Garcinia multiflora, were found to be effective in inhibiting viral activity and may be used in a method for treating and/or preventing a broad range of viral infections such as Influenza A and B, hepatitis B and HIV-1, HSV-1, HSV-2, VZV, and measles. It has been discovered that robustaflavone effectively inhibits activity of influenza A and B viruses, hepatitis B, HIV-1, HSV-1 and HSV-2. Hinokiflavone and morelloflavone exhibited similar activity against various strains of HIV-1.
Anti-viral biflavanoid derivatives and salts and pharmaceutical compositions containing the same are also contemplated by the invention. Representative derivatives include ethers, e.g., methyl ethers, esters, amines, ethylene oxide adducts, and polymers such as trimers and tetramers of apigenin. Representative salts include sulfates and acid salts. Methods for preparing these derivatives and salts are also provided. It has been discovered for instance that salts of robustaflavone, e.g., robustaflavone tetrasulfate potassium salt and robustaflavone tetrasodium salt, effectively inhibit hepatitis B activity. Furthermore, robustaflavone hexa-O-acetate and hexa-O-methyl ether derivatives of robustaflavone were found to be not only potent inhibitors of HBV replication but with greatly reduced cytotoxicity. Scheme I illustrates several examples of biflavanoid derivatives.
Improved methods for extracting robustaflavone from plant material are also provided. According to one method, a substantially pure robustaflavone in greater yields can be obtained through the use of a particular solvent mixture comprising toluene/ethanol/pyridine. The improved extraction method eliminates the use of benzene and requires smaller volumes of pyridine from the prior reported methods.
A second improved method for purification of robustaflavone is also provided which involves acetylation of the extracted pigment to produce acetates of the pigments. Robustaflavone acetate is then purified by recrystallization and converted to robustaflavone by hydrolysis. This method eliminates the use of pyridine in column chromatography and is ideal for large scale extraction of robustaflavone.
Finally, a method for treating and/or preventing viral infections using antiviral biflavanoids alone or in combination with one or more other antiviral agents is described. Representative viral infections include influenza A and B viruses, hepatitis B and human immunodeficiency virus (HIV-1), HSV-1, HSV-2, VZV, and measles.
Accordingly, it is an object of the invention to provide substantially purified antiviral biflavanoids robustaflavone, hinokiflavone, amentoflavone, agathisflavone, morelloflavone, rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a.
It is another object of the invention to provide antiviral derivatives and salt forms of biflavanoids robustaflavone, hinokiflavone, amentoflavone, agathisflavone, morelloflavone, volkensiflavone, rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a as well as method of preparation thereof. A representative example of an antiviral biflavanoid derivatives include robustaflavone tetrasulfate potassium salt, robustaflavone tetrasodium salt, robustaflavone hexa-O-acetate, and robustaflavone hexa-O-methyl ether.
It is yet another object of the invention to provide pharmaceutical compositions which include at least one antiviral biflavanoids such as robustaflavone, hinokiflavone, amentoflavone, agathisflavone, morelloflavone, volkensiflavone, rhusflavanone, succedaneaflavanone, GB-1a, GB-2a, derivatives or salts thereof. Pharmaceutical compositions including an effective amount of at least one antiviral biflavanoid in combination with at least one other antiviral agent, e.g. robustaflavone with penciclovar or lamivudine (3TC), for use in combination antiviral therapy are also contemplated.
It is a further object of the invention to provide an improved method for obtaining substantially pure robustaflavone and in greater yields than prior procedures.
It is yet a further object of the invention to provide a method for treating and/or preventing viral infections which comprises administering an antiviral effective amount of a biflavanoid. Representative viral infections are caused by viral agents such as influenza, e.g., influenza A and B; hepatitis, e.g., hepatitis B; human immunodeficiency virus, e.g., HIV-1; HSV-1, HSV-2, VZV, and measles.
These and other objects of the invention will become apparent in light of the detailed description below. 